The concept of gene therapy is based on that nucleic acids, DNA, RNA can be used as pharmaceutical products to cause in vivo production of therapeutic proteins at appropriate sites. Delivery systems for nucleic acids are often classified as viral and non-viral delivery systems. Because of their highly evolved and specialized components, viral systems are currently the most effective means of DNA delivery, achieving high efficiencies for both delivery and expression. However, there are safety concerns for viral delivery systems. The toxicity, immunogenicity, restricted targeting to specific cell types, limited DNA carrying capacity, production and packaging problems, recombination and a very high production cost hamper their clinical use (Luo and Saltzman, 2000). For these reasons, non-viral delivery systems have become increasingly desirable in both basic research laboratories and clinical settings. However, from a pharmaceutical point of view, the way of delivery of nucleic acids still remains a challenge since a relatively low expression is obtained in vivo with non-viral delivery systems as compared to viral delivery systems (Saeki et al., 1997).
A variety of non-viral delivery systems, including cationic lipids, peptides or polymers in complex with plasmid DNA (pDNA), have been described in the prior art (Boussif et al., 1995; Felgner et al., 1994; Hudde et al., 1999). The negatively charged nucleic acids interacts with the cationic molecules mainly through ion-ion interactions, and undergo a transition from a free form to a compacted state. In this state the cationic molecules may provide protection against nuclease degradation and may also give the nucleic acid-cationic molecule complex surface properties that favour their interaction with and uptake by the cells (Ledley, 1996).
Among these cationic molecules, the synthetic polymer polyethylenimine (PEI) have been shown to form stable complexes with pDNA and mediate relatively high expression of the transgene both in vitro and in vivo (Boussif et al., 1995; Ferrari et al., 1997; Gautam et al., 2001). For this reason, PEI is often used as a reference system in the experimental setup. However, a rough correlation between toxicity and efficiency has been suggested for PEI (Luo and Saltzman, 2000) and recent studies have addressed concerns about toxicity using PEI (Godbey et al., 2001; Putnam et al., 2001). Another drawback with PEI is that it is not biodegradable and it may therefore be stored in the body for a long time. Therefore, the search for effective and non-toxic biodegradable non-viral delivery systems is highly desirable.
Most commonly, non-viral delivery systems have been delivered in vivo by the parenteral route. After intravenous administration to mice, compacted nucleic acid-cationic molecule complexes deposited mainly in the lung capillaries where the gene was expressed in the endothelium of the capillaries in the alveolar septi (Li and Huang, 1997; Li et al., 2000; Song et al., 1997) or even in the alveolar cells (Bragonzi et al., 2000; Griesenbach et al., 1998), but not in the epithelium. However, unformulated, naked DNA was rapidly degraded in the blood circulation before it reached its target and generally resulted in no gene expression. In contrast, injection of naked DNA into skeletal muscle resulted in a dose-dependent gene expression (Wolff et al., 1990) which was further enhanced when complexed with a non-compacting but ‘interactive’ polymer such as polyvinyl pyrrolidone (PVP) or polyvinyl alcohol (PVA) (WO 9621470) (Mumper et al., 1996; Mumper et al., 1998). Thus, gene transfection in vivo is tissue-dependent in an unpredictable way and therefore remains a challenge.
Mucosal delivery of non-viral delivery systems has also been described that is delivery to the gastrointestinal tract, nose and respiratory tract (Koping-Hoggard et al., 2001; Roy et al., 1999), WO 01/41810. With exception for the delivery to the nasal tissue where DNA in uncompacted form gives the best gene expression (WO 01/41810) compacted nucleic acid-cationic molecule complexes are preferred to uncompacted DNA when a high gene expression is required in a mucosal tissue.
In prior art, non-viral gene delivery systems are based on cationic polymers such as chitosan of rather high molecular weight, often several hundred kilodaltons (kDa) with 5 kDa as a lower limit, see for example MacLaughlin et al., 1998, Roy et al., 1999 and WO 97/42975. The major reason is that polymers of lower molecular weight (<5 kDa) form unstable complexes with DNA, resulting in a low gene expression (Koping-Hoggard, 2001). However, there are many drawbacks using cations of high molecular weight such as increased aggregation of compacted nucleic acid-cationic molecule complexes and solubility problems (MacLaughlin et al., 1998). Further, there are several biological advantages of using cationic molecules of lower molecular weights that is they generally show reduced toxicity and reduced complement activation compared to cations of higher molecular weights (Fischer et al., 1999; Plank et al., 1999).
In the prior art some examples of the use of low molecular weight cations for complexation with nucleic acid have been described (Florea 2001; Godbey et al., 1999; Koping-Hoggard, 2001; MacLaughlin, et al., 1998; Sato et al., 2001). However, these low molecular weight cations form unstable compacts with DNA that separate in an electric field (agarose gel electrophoresis) resulting in no or a very low gene expression in vitro, as compared to cations of higher molecular weights. This can be explained by that complexes formed between DNA and low molecular weight cations are generally unstable and dissociate easily (Koping-Hoggard, 2001). In fact, the dissociation of cationic molecule-DNA compacts and release of naked DNA during agarose gel electrophoresis has often been used as an assay to distinguish ineffective formulations from effective ones in the literature (Fischer et al., 1999; Gebhart and Kabanov, 2001; Koping-Hoggard et al., 2001). Then, it is known from the prior art that complexes between DNA and cations should be stable to mediate a high gene expression.
The prior art contains various examples of methods for the delivery of nucleic acids to the respiratory tract using non-viral vectors (Deshpande et al., 1998; Ferrari et al., 1997; Gautam et al., 2000). We recently identified and characterized one such system based on the DNA-complexing polymer chitosan (Koping-Hoggard et al., 2001), a linear polysaccharide, which can be derived from chitin. Chitosan-based gene delivery systems are also described in U.S. Pat. No. 5,972,707 (Roy et al., 1999), WO 98/01160 and in US patent application no. 2001/0031497 (Rolland et al., 2001).
Chitosan has been introduced as a tight junction-modifying agent for improved drug delivery across epithelial barriers (Artursson et al., 1994). It is considered to be non-toxic after oral administration to humans and has been approved as a food additive and also incorporated into a wound-healing product (Illum, 1998).
Chitosans comprise a family of water-soluble, linear polysaccharides consisting of (1→4)-linked 2-acetamido-2-deoxy-β-D-glucose (GlcNAc, A-unit) and 2-amino-2-deoxy-β-D-glucose, (GlcN, D-unit) in varying composition and sequence (FIG. 1). The definition adopted here to distinguish between chitin and chitosan is based on the insolubility of chitin in dilute acid solution and the solubility of chitosan in the same dilute acid solution (Roberts, 1992).
The relative content of A- and D-units may be expressed as the fraction of A-units:
FA=number of A-units/(number of A-units+number of D-units)
FA is related to the percentage of de-N-acetylated units through the relation:
% de-N-acetylated units=100%·(1−FA)
Each D-unit contains a hydrophilic and protonizable amino group, whereas each A-unit contains a hydrophobic acetyl group. The relative amounts of the two monomers (that is A/D=FA/(1−FA)) can be varied over a wide range, and results in a broad variability in their chemical, physical and biological properties. This includes the properties of the chitosans in solution, in the gel state and in the solid state, as well as their interactions with other molecules, cells and other biological and non-biological matter.
The influence of the chemical structure of chitosans was recently demonstrated when chitosans were used in a non-viral gene delivery system (Koping-Hoggard et al., 2001). Chitosans of different chemical compositions displayed a structure dependent efficiency as gene delivery system. Only chitosans that formed stable complexes with pDNA gave a significant transgene expression. Such complexes required that at least 65% of the chitosan monomers were deacetylated.
Chitosans can be depolymerized either chemically or enzymatically to obtain chitosan polymers or oligomers of the desired molecular size. Various chemical degradation mechanisms can be used to depolymerize chitosans, that is acid hydrolysis, nitrous acid and oxidative-reductive depolymerization. Ultrasonic depolymerisation of polymers may alternatively be used, but these methods are very inconvenient for producing very low molecular weights. Depolymerisation of chitosan by the use of nitrous acid is a convenient way of preparing low-molecular weight chitosan, as described in for example U.S. Pat. No. 3,922,260 and U.S. Pat. No. 5,312,908. This mechanism involves deamination of a D-unit, forming 2,5-anhydro-D-mannose unit at the new reducing end, which can be reduced to 2,5-anhydro-D-mannitol using NaBH4 as shown in FIG. 2. Alternatively, various enzymes can also be used to depolymerize chitosan, for instance U.S. Pat. No. 5,482,843, chitosanases, chitinases, and lysozyme. Also acid hydrolysis may be used to depolymerise chitosan (Vårum et al., 2001, and references therein).
In the prior art, studies of the effect of molecular weight of chitosan on transfection efficiency in vitro of chitosan-pDNA complexes showed no significant dependence of the molecular weight in the size range 20-200 kDa (Koping-Hoggard et al., 2001; MacLaughlin et al., 1998). However, in another study (Sato et al., 2001) chitosans of 15 kDa and 52 kDa showed higher gene expression than chitosan>100 kDa, while no gene expression was detected with a 1.3 kDa chitosan. Further, studies of gene expression in vitro and in lung tissue in vivo using a series of low molecular weight chitosans (1.2 kDa, 2.4 kDa and 4.7 kDa) showed that only the 4.7 kDa chitosan mediated a significant gene expression (Koping-Hoggard, 2001).
Chitosans of different molecular weights have been used as components in complexes for non-viral gene delivery. For example, US patent application no. 2001/0031497A refers to the use of small molecular weight chitosan as a component of the delivery system, that is chitosan in the range of 2-4 kDa Mw, which resulted in the smallest particle of gene delivery system and also in an increased transfection of cells with the condensed delivery system in vitro.
Chitosans of different molecular weights which are used in gene delivery systems are normally unfractionated samples obtained from commercial suppliers, and lower molecular weights are obtained from said samples by partial degradation using degradation agents such as organic or inorganic acids, nitric acid or chitosan degrading enzymes. In all cases, the distribution of molecular weights remains relatively high. As an example, a commercial chitosan with a weight average molecular weight (Mw) of 180.000 was analysed by size-exclusion chromatography using a refractive index detector and a multi-angle laser light scattering detector. FIG. 3A shows the elution profile, that is refractive index detector signal, which is proportional to the concentration of chitosan, combined with a plot of the calculated molecular weight (expressed as chitosan in the acetate salt form) as a function of the elution volume. It is evident that the sample contains molecular weights as high as 106 g/mol (1000 kDa) at the beginning of the peak and as low as 104 (10 kDa) at the end of the peak. A recalculation of these data gives the cumulative molecular weight distribution (FIG. 3B). It may be inferred from these calculations that 12% (w/w) of the sample has a molecular weight below 40 kDa and 38% of the sample has a molecular weight below 100 kDa. Likewise, 18% of the sample has a molecular weight above 300 kDa and 9% has a molecular weight above 400 kDa. The sample is thus polydisperse since it contains polymers of different molecular weights or chain lengths.
Chitosans may be supplied in the free amine form or as different salts such as chitosan chloride, chitosan glutamate and chitosan acetate. The salt-form influences the relationship between the molecular weight (M) and DP (the number of sugar residues per molecule). The following equations describe this relationship between DP and M:
Free base: M=DP (161(1−FA)+203FA)=DP (161+42FA)
Chitosan chloride: M=DP (197.45(1−FA)+203FA)=DP (197.45+5.55FA)
Chitosan acetate: M=DP (221(1−FA)+203FA)=DP (221−18FA)
Chitosan glutamate: M=DP (308(1−FA)+203FA)=DP (308−105FA)
The weight average molecular weight (Mw) of a polydisperse sample may be expressed as Mw=ΣciMi/Σci where ci is the concentration (g/l) of a particular molecular weight (Mi) within the distribution) (Tanford, C. (1961) Physical chemistry of macromolecules, John Wiley and Sons, New York, Section 8b). Likewise, the number average molecular weight (Mn) may be expressed as Mn=Σci/Σ(ci/Mi). In the case referred to above Mw=180 kDa and Mn=84.5 kDa, and the polydispersity index which is defined as Mw/Mn equals 2.1. A polydispersity near 2 is characteristic of a linear polymer which has been subjected to random depolymerisation (Tanford, C. (1961) Physical chemistry of macromolecules, John Wiley and Sons, New York, Section 33a)
The distribution of chain lengths following a random depolymerisation of a linear polymer such as chitosan is given by the equation (Tanford (1961):Wx=xpx−1(1−p)2 Wx is the weight fraction of chains containing x monomers (for chitosan the monomers are sugar residues) and p is the fraction of intact linkages and 1−p is the fraction of cleaved linkages. The number average degree of polymerisation (xn) equals 1/(1−p). Since Mn=M0xn, where M0 is the monomer equivalent weight, which is 203 g/mol for a residue of N-acetyl-glucosamine when it occurs within a chitosan chain and 161 g/mol for a residue of glucosamine in the free base form when it occurs within a chitosan chain. For a given FA the average M0 becomes equal to 203·FA+161·(1−FA).
FIG. 4 shows SEC-MALLS chromatograms (4A), and differential (4B) and cumulative (4C) molecular weight distributions of a chitosan, which has been depolymerised by nitrous acid to obtain different weight average molecular weights in the range from 41.500 to 13.400. It is clearly shown that the calculated molecular weight distributions remain broad. These data clearly demonstrate that chitosans of different molecular weights which are produced from a high molecular weight by partial degradation remain polydiserse and contain chains of widely differing molecular weights.
The molecular weight distribution of a polymer may be modified by selectively removing certain parts of the distribution. Chitosan samples with relatively short chains may be fractionated by gel filtration to obtain individual oligomers or fractions with relatively narrow molecular weight distributions. One example is given by Tøommeraas et al. (2001) who obtained purified chitosan oligomers in the range of 2-10 residues per chain.
Samples with higher molecular weights may also be fractionated by gel filtration as demonstrated for chitosans by Ottøy et al. (1996). Typically, fractions with Mw/Mn values of 1.2-1.5 was obtained by fractionating a normally polydisperse sample with Mw=270.000 using a gel filtration column containing Sepharose CL-4B and Sepharose CL-6B.
In an alternative method polydisperse chitosans may be fractionated by dialysis or membrane techniques which allow selective removal of the shortest chains, and where the resulting distribution depends on the initial distribution as well as the membrane characteristics porosity and transport coefficients and the operating conditions.
According to the present invention it was surprisingly discovered that chitosans of a single chain length or chitosans with narrow molecular weight distributions had different properties as complexing agents in gene delivery than other samples of comparable Mw or Mn, but with broader molecular weight distributions.
Another disadvantage of many cations used for complexation of nucleic acid e.g. PEI, polylysine and chitosan is that they are roughly processed bulk chemicals with a broad molecular weight distribution and hence rather undefined (Godbey et al., 1999). It is well established that such chemicals may display a batch to batch variation. Therefore, from a pharmaceutical point of view, well-defined polycations having a narrow molecular weight distribution are preferred.
Another disadvantage using broad molecular weight polycations for complexation of nucleic acids and subsequent transfection is that chains of differents lengths may have different complexation and transfection effectivities.